Sniffer cells for the detection of neural Angiotensin II in vitro

Abstract

Neuropeptide release in the brain has traditionally been difficult to observe. Existing methods lack temporal and spatial resolution that is consistent with the function and size of neurons. We use cultured "sniffer cells" to improve the temporal and spatial resolution of observing neuropeptide release. Sniffer cells were created by stably transfecting Chinese Hamster Ovary (CHO) cells with plasmids encoding the rat angiotensin type 1a receptor and a genetically encoded Ca2+ sensor. Isolated, cultured sniffer cells showed dose-dependent increases in fluorescence in response to exogenously applied angiotensin II and III, but not other common neurotransmitters. Sniffer cells placed on the median preoptic nucleus (a presumptive site of angiotensin release) displayed spontaneous activity and evoked responses to either electrical or optogenetic stimulation of the subfornical organ. Stable sniffer cell lines could be a viable method for detecting neuropeptide release in vitro, while still being able to distinguish differences in neuropeptide concentration.

Figure 1

Cropped Western blots showing selection of sniffer cells - stably transfected CHO cell colonies expressing high levels of (A) GCaMP + AT1aR, (B) GCaMP only, and (C) R-GECO + AT1aR (see Supplemental Information for full Western blots). Optimally expressed colonies (bolded and underlined) selected: 1, 3, and 10 for GCaMP + AT1aR; 1, 10, and 11 for GCaMP only; and 8 and 11 for R-GECO + AT1aR. Sniffer cells exhibit variable baseline fluorescence. (D) GCaMP + AT1aR sniffer cells plated on coverslips exhibit variable levels of GCaMP fluorescence. (E) The variability in sniffer cell fluorescence is not different between CHO cells transfected with GCaMP or GCaMP + AT1aR. (F) Sniffer cells plated on coverslips exhibit variable levels of R-GECO fluorescence. (G) The variability in sniffer cell fluorescence is not different between CHO cells transfected with R-GECO or R-GECO + AT1aR. F-Test **p < 0.01.

Figure 2

ANG II mediated increases in sniffer cell fluorescence. (A) ANG II (100 nM) induced a robust but transient increase in GCaMP fluorescence that was blocked by the AT1aR receptor antagonist Losartan (10 µM). Control n = 39, Losartan n = 38. (B) Data shows that bath application of glutamate (50 µM), GABA (50 µM), and carbachol (50 µM) failed to change fluorescent intensity of sniffer cells transfected with GCaMP (n = 29) or GCaMP + AT1aR (n = 38). ANG II (100 nM) did increase fluorescent intensity of sniffer cells, but only in sniffer cells transfected with GCaMP + AT1aR. Dose-dependent effects of ANG II and related compounds were also measured. (C) GCaMP + AT1aR sniffer cells exhibit dose-dependent increases in fluorescence in response to bath application of ANG II (100 nM, n = 10). (D) Bath application of ANG III induced a dose-dependent increase in GCaMP + AT1aR sniffer cell fluorescence. Bath application of ANG (1–7) or bradykinin did not induce a change in GCaMP + AT1aR sniffer cell fluorescence at any of the doses tested (0.1–100 nM, n = 17–41). (E) R-GECO + AT1aR sniffer cells exhibit dose-dependent increases in fluorescence in response to bath application of ANG II (n = 17). R-GECO only cells did not respond to ANG II (n = 27). (F) ANG II-mediated increases in R-GECO + AT1aR are blocked by bath application of Losartan (10 µM, n = 17). *p < 0.05, **p < 0.01.

Figure 3

Sniffer cells exhibit spontaneous increases in fluorescence on in vitro brain slices. (A) GCaMP + AT1aR sniffer cells placed on the MnPO exhibit spontaneous increases in fluorescence that are completely blocked by bath application of Losartan (10 µM). (B) Spontaneous sniffer cell responses return following washout of bath applied Losartan. (C) Bath application of TTX (1 µM) was also effective in reducing the number of spontaneous sniffer cell responses. (D) R-GECO + AT1aR sniffer cells also exhibit spontaneous increases in fluorescence on in vitro brain slices. Each trace represents an individual sniffer cell.

Figure 4

Sniffer cells respond to both electrically and optogenetically evoked ANG II release. (A) The SFO was electrically stimulated (arrow) and evoked release of AT1aR agonist that was detected in the MnPO. (B) The SFO was stimulated with 590 nm light and evoked release of AT1aR agonist that was detected in the MnPO with GCaMP + AT1aR sniffer cells. The light evoked sniffer cell response was reproducible. (C) Summary of GCaMP + AT1aR sniffer cell responses from in vitro slices. Optogenetic stimulation produced responses similar in magnitude to spontaneous events. Electrical stimulation produced responses that were greater than both light-evoked or spontaneous responses. (D) The SFO was stimulated with 470 nm light and evoked release of AT1aR agonist that was detected in the MnPO with R-GECO + AT1aR sniffer cells. *p 0.05.